WO2021133272A2

WO2021133272A2 - Lycopene extraction from watermelon by using non-toxic eco-friendly materials

Info

Publication number: WO2021133272A2
Application number: PCT/TR2019/051219
Authority: WO
Inventors: Asiye AKYILDIZ; Erdal AGCAM; Nuray INAN CINKIR
Original assignee: Cukurova Universitesi Rektorlugu
Priority date: 2019-12-23
Filing date: 2019-12-26
Publication date: 2021-07-01
Also published as: WO2021133272A3

Abstract

The present invention relates to lycopene extraction from the watermelon using surfactants (Span 20 and sucrose monopalmitate) and co-surfactants (glycerol and NaCI).

Description

LYCOPENE EXTRACTION FROM WATERMELON BY USING NON-TOXIC ECO-

FRIENDLY MATERIALS TECHNICAL FIELD

The present invention relates to lycopene extraction from the watermelon using surfactants (Span 20 and sucrose monopalm itate) and co-surfactants (glycerol and NaCI). PRIOR ART

Lycopene as an unsaturated hydrocarbon containing 13 pairs of bonds in the carotenoid group which is a bioactive component responsible for the red colour in fruits and vegetables and is oil-soluble. Recently, lycopene has attracted attention as a pharmacological component. Lycopene has biological activities such as antioxidant activity, induction of inter-cell communication, control of growth, and protection of the skin against UV radiation. It is recommended as an alternative to synthetic food colouring agents, as well as potential health benefits.

Commercial lycopene can be derived from tomato by solvent extraction (such as hexane, acetone, methanol, chloroform, petroleum ether, ethyl acetate) or chemically synthesized. In recent years, consumer trends have led to natural products and bioactive components extracted from them. Tomatoes are used commercially as the most important source of natural lycopene. Other important sources of lycopene, such as watermelon, are not commercially evaluated. In addition, the commercial lycopene has a high unit price. Watermelon is an important alternative resource for increasing commercial lycopene resources and lowering the lycopene unit price. Besides the fresh consumption of the watermelon, a new area having high economic value will be created for manufacturers, which will also be evaluated in commercial lycopene production.

Until now, studies on lycopene extraction have focused mainly on tomato and tomato products. In this context, studies on the evaluation of other lycopene sources except tomatoes are limited. In previous studies, lycopene extraction is mainly carried out by the solvent extraction. The effect of non-thermal technologies on lycopene extraction has been determined in recent periods. However, these techniques (ultrasonic-assisted extraction, microwave-assisted extraction, l supercritical fluid extraction, accelerated pressure extraction, etc.) are not suitable for industrial production due to the need for high energy and expensive equipment. In addition, microbial lycopene production is carried out using microorganisms such as Blakeslea trisport and Rhodospirillum rubrum. However, lycopene production with this method is limited to the laboratory scale and lycopene yield is very low.

Hexane and its mixtures are used as solvents in the extraction of carotenoids. The earlier studies showed that nervous disorders are detected in people exposed to 125 ppm daily for 3 months. In addition, hexane is an explosive material. Therefore, our main objective in lycopene extraction is to use environmentally friendly non- thermal extraction techniques that reduce extraction time, solvent consumption and increase extraction efficiency at the lowest possible cost.

Surfactants are seen as a good solution to the problems mentioned above. Because there is no need for expensive equipment during their application. Significant energy savings are also available. The use of organic solvents is not needed. Non-ionic surfactants as emulsifiers are preferred in the food industry. Non ionic surfactants are not classified as toxic compounds. It is not necessary to separate the surfactant from the extract after extraction. Thus, the process cost is significantly reduced. Non-ionic surfactants (Span 20, Span 40, Span 85, Tween 20, Tween 60, Tween 80, saponin, rhamnolipid, sucrose monopalm itate, Triton X-100 and lecithin) are accepted as edible and non-toxic by FDA (Food and Drug Administration).

BRIEF DESCRIPTION OF THE INVENTION

The method of the present invention is described the extraction of lycopene from watermelon pulp by means of microemulsion technique using surfactants that are allowed to be used in foods instead of solvents such as hexane, acetone, ether, methanol, chloroform which are known to have adverse effects on environmental and health. In addition, this method has a significant contribution potential to the country's economy, consumer health, sustainable green approach and food industry by converting to high added products such as high-purity commercial lycopene other than the fresh consumption of the watermelon with significant production in Turkey and having low processing possibility in food industry. It is possible to recover hydrophilic and hydrophobic bioactive components from foods by changing the colloid structure of the matrix using surfactants. From this point, in the present invention, lycopene is extracted from the watermelon by using non-ionic surfactants instead of solvents with high toxicity potential. In our method, lycopene is taken from the structure by protecting against environmental factors. Because sheath is formed around the lycopene by surfactants. The surfactants are suitable for use in foods and are edible. There is no need to remove them from the structure. Thus, the process cost is reduced. In addition, experimental designs are designed for the most suitable surfactants and co-surfactants determined by the research, and extraction conditions are optimized for the highest lycopene efficiency by means of the obtained mathematical equations.

LIST OF THE FIGURES

Figure 1. Flow diagram of the lycopene extraction method with the microemulsion technique according to the conditions of extraction-1 (Span 20- Glycerol) Figure 2. Flow diagram of the lycopene extraction method with the microemulsion technique according to the conditions of extraction-2 (Sucrose monopalmitate-NaCI)

DETAILED DESCRIPTION OF THE INVENTION The seeds and peels portions of the watermelons are removed and the edible parts are pulped in the high speed blender as soon as possible to prevent lycopene degradation with minimal interaction with O2 and light. The pulp samples are concentrated approximately up to 30% dry matter content at 55°C in the rotary vacuum evaporator. In this way, watermelon pulp is also used possible during periods when watermelon is not grown, and storage area is also saved. The watermelon pulp is not applied to any process for improving extraction efficiency. The concentrated samples are stored at -60°C until other processing steps are performed.

In order to develop the method of the present invention, non-ionic surfactants such as Span 20 (HLB:8.6), Tween 60 (HLB:14.9) sucrose monopalmitate (HLB:15) and saponin (HLB:13.5) and co-surfactants such as glycerol, propylene glycol (propanediol), ethanol, NaCI are used. In preliminary trials, each surfactant and co surfactants are matched together. The non-ionic surfactant and co-surfactants pairs with high extraction efficiency are determined as Span 20-Glycerol and Sucrose monopalmitate (SM)-NaCI. Extraction conditions containing Span 20-Glycerol are considered as Extraction-1 and extraction conditions containing SM-NaCI are considered as Extraction-2.

Preliminary Trial Conditions for Extraction-1: The mixture (A) of 0.5 g of watermelon pulp and 9.5 g of glycerol is mixed in the magnetic stirrer for 2 minutes, and added Span 20 (w/v) corresponding to 10-20% of phase A. Then, it is mixed again in the magnetic stirrer for 5 minutes.

Preliminary Trial Conditions for Extraction-2: The mixture (B) of 0.5 g of watermelon pulp and 9.5 ml_ of ultra-pure water is mixed with NaCI (w/v) (up to 20- 35% of phase (B) in a magnetic stirrer for 2 minutes. Sucrose monopalmitate (w/v) corresponding to 5% of Phase B is added and mixed again in the magnetic stirrer for 5 minutes.

The homogeneous mixtures obtained after the application of the conditions defined above for Extraction-1 and Extraction-2 are kept in the water bath at 55°C for 30 minutes. After the equilibration procedure, the mixtures are centrifuged at 6000 rpm and 4°C for 20 minutes and upper phase (surfactant rich phase) is viscous. The rest (sub-phase) is the aqueous phase.

During the preliminary trials of the Extraction-1 subject to the present invention, at least one of the glycerol, propylene glycol and ethanol such as hydrophilic materials could be used as aqueous phase instead of water.

Optimization

Experimental designs for the optimization study are formed by using the preliminary trials results relates to preliminary trails Extraction-1 and Extraction-2. Independent variables for Extraction-1: Span 20/sample amount (0.2-8 g/g), glycerol/sample amount (10-20 g/g), water bath temperature (25-80°C) and incubation time (5-60 minutes); independent variables for Extraction-2: SM/sample amount (0.2-2 g/g), NaCI/sample amount (0.2-10 g/g), water bath temperature (25- 80°C) and incubation time (5-60 minutes). Dependent variable for both extraction conditions (1 and 2) is the amount of lycopene obtained with HPLC analysis.

For the optimization study mentioned above, the independent variables change Span-20 1-40% of the phase A, glycerol 5-10 g for Extraction-1 ; SM 1-10% of phase B, NaCI 1-50% of the phase B for Extraction-2. The amount of watermelon pulp is fixed and used as 0.5 g during laboratory-scaled studies. The optimization study is performed depending on the sample quantity. Thus, when the amount of sample increases, optimum conditions can be applied proportionally. In the optimization studies, optimum conditions are determined for both extraction methods. Optimized Extraction-1: 0.5 g of watermelon pulp and 4.4 g of glycerol are mixed for 2 minutes in a 50 mL of the beaker and then added 3.01 g of surfactant (Span 20 (HLB:8.6)) and mixed again in the magnetic stirrer for 5 minutes for achieving uniform dispersion. The stirring time in the magnetic stirrer is determined by preliminary tests. Then, the mixture is transferred to a centrifuge tube and the tube is kept in a water bath at 38.8°C for 18.8 min. After the incubation, the mixture is centrifuged (6000 rpm, 20 min, 4°C) (Figure 1). The upper phase (surfactant rich phase) is viscous. The rest (sub-phase) is the aqueous phase.

Optimized Extraction-2: 0.5 g of watermelon pulp, 9.5 mL of ultra-pure water and 3.76 g of NaCI are mixed in the magnetic stirrer in the beaker for 2 minutes and then added 0.57 g of surfactant (Sucrose monopalm itate) (HLB:15) and mixed again in the magnetic stirrer for 5 minutes for achieving uniform dispersion. The stirring time in the magnetic stirrer is determined by preliminary tests. Then, the mixture is transferred to a centrifuge tube and the tube is kept in a water bath at 66.3°C for 40.8 min. After the incubation, the mixture is centrifuged (6000 rpm, 20 min, 4°C) (Figure 2). The upper phase (surfactant rich phase) is viscous. The rest (sub-phase) is the aqueous phase.

Based on optimum extraction conditions, the lycopene yield in the collected extracts for Extraction-1 and Extraction-2 are > 96,5% and > 83,6%, respectively. Microemulsions have high selectivity and solubility for oil-soluble and water- soluble components because of nanostructures with different degrees of curvature and characteristics within the same phase. Due to this feature, oil and water-soluble components can be extracted simultaneously. Microemulsion systems are used in many areas, such as pharmaceuticals, cosmetics and food. Microemulsions in the field of pharmaceuticals and cosmetics are common because they improve the solubility and bioavailability of hydrophobic components. There are many bioactive components in various foods that positively affect the health. These bioactive components generally show poor solubility and low bioavailability. In addition, these components are sensitive to external factors and lost their effectiveness by degradation. Microemulsions, a carrier system, protect lipophilic bioactive components against oxidation, enzymatic hydrolysis and increasing bioavailability by improving their resolution. Surfactants identified within the scope of the present invention:

Lipophilic sorbitan fatty acid esters (Span 20, sorbitan monolaurate) are obtained from vegetable and animal-origin lauric acid and sorbitol. The non-ionic surfactant is used to form water in oil emulsions. Another surfactant, sucrose monopalm itate, is increasingly used in the food and pharmaceutical sectors. Sucrose monopalm itate as natural product is obtained from sucrose and vegetable oils. Sucrose monopalm itate is a non-ionic surfactant having one fatty acid as hydrophobic tail and sucrose as hydrophilic head group. It is hydrophilic and has a high HLB value (hydrophilic-lipophilic balance) and stabilizes oil in water emulsions. Compared to synthetic surfactants, it is preferred due to their low toxic effects, eco- friendly, high bioavailability and good taste-aroma profile.

For the Extraction-1 and Extraction-2 method subject to the present invention;

- The type and matching levels of surfactants and co-surfactants and the balance of the proportions used in the extraction, - The stirring time is important to form an uniform emulsion system from the surfactant and co-surfactant, water and watermelon pulp,

- An adequate level of temperature and incubation time of the water bath,

- The centrifugation at high speed and low temperature are applied to ensure phase separation.

Claims

1. A method of lycopene extraction from watermelon characterized by the steps of;

- The seeds and peels portions of the watermelons are removed and the edible parts are pulped in the high speed blender as soon as possible to prevent lycopene degradation with minimal interaction with O2 and light,

- adding 4.4 g of glycerol and 0.5 g of watermelon pulp,

- Homogenized by mixing in a magnetic stirrer for 2 minutes in a 50 mL beaker,

- Addition of 3.01 g of surfactant (Span 20 (HLB:8.6)),

- Stirring the mixture for 5 minutes in a magnetic stirrer,

- After the mixture is transferred to a centrifuge tube, which is waited in a water bath at 38.8°C for 18.8 min.,

- After the incubation, mixture is centrifuged (6000 rpm, 20 min, 4°C).

2. A method of lycopene extraction from watermelon characterized by the steps of;

- Mixing 0.5 g of watermelon pulp, 9.5 mL of ultra-pure water and 3.76 g of NaCI in the magnetic stirrer in the 50 mL beaker for 2 minutes,

- Addition of 0.57 g of surfactant (sucrose monopalmitate (HLB: 15)),

- Stirring the mixture for 5 minutes in a magnetic stirrer,

- After the mixture is transferred to a centrifuge tube, which is waited in a water bath at 66.3°C for 40.8 min,

- After the incubation, mixture is centrifuged (6000 rpm, 20 min, 4°C).